The long term goal of this project is to define the molecular interactions of cocaine with dopaminergic systems. Many of the behavioral effects of cocaine are attributed to its ability to block the reuptake of dopamine in mesolimbic neurons. Consequently, increased extracellular dopamine may enhance postsynaptic transmission and/or modulate presynaptic dopamine receptors known to inhibit neurotransmitter synthesis as well as further release. The specific aim of this application is to dissect this system by focussing on presynaptic events associated with cocaine's purported modulation of dopamine autoreceptors. Towards this end, model neuronal systems have been engineered by transfecting immortalized mesencephalic dopamine producing cell lines with cloned D2 and D3 receptors. While traditionally D2 receptors have been implicated in autoreceptor function, it is not known which of the D2-like subtypes subserves autoregulation of release and/or synthesis. To test the hypothesis that the D2-like receptors subserve different roles, the transfected cell lines will be systematically tested for receptor mediated effects on synthesis and release. Preliminary results show that agonist stimulation ofD2 and D3 receptors lead to subtype specific reductions in dopamine release and synthesis. These data imply specific roles for each receptor and suggest the effects of cocaine may vary depending upon receptor subtype. Studies outlined in this proposal will l) determine the effect of various D2 agonists on dopamine release in the individual transfected cell lines as well as the general roles of pertussis toxin, cation channels, desensitization and phosphorylation pathways in modulating this response. 2) Determine the role of receptor stimulation and desensitization on dopamine synthesis by measuring tyrosine hydroxylase activity and changes in the state of tyrosine hydroxylase phosphorylation. The roles of various signal transduction pathways will be tested by using specific protein kinase and phosphatase inhibitors to alter autoregulation of dopamine synthesis. In either case receptor coupling to specific GTP binding proteins will be tested for using mutant G proteins. 3) Test the effect of acute and chronic cocaine on autoreceptor function in both the release and synthesis paradigms. These studies have direct implications for understanding the presynaptic mechanisms involved in the reinforcing and behavior sensitization effects of cocaine.